Injectable chitosan mixtures forming hydrogels

ABSTRACT

A chitosan composition which forms a hydrogel at near physiological pH and 37° C., comprising at least one type of chitosan having a degree of acetylation in the range of from about 30% to about 60%, and at least one type of chitosan having a degree of deacetylation of at least about 70% is disclosed. Further disclosed is a chitosan composition which forms a hydrogel at near physiological pH and 37° C., that includes at least one type of chitosan having a degree of deacetylation of at least about 70% and a molecular weight of from 10-4000 kDa, and at least one type of a chitosan having a molecular weight of from 200-20000 Da. Further disclosed are methods of preparation and uses of the chitosan compositions.

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, relates topositively charged polysaccharide hydrogels, and, more particularly, topH-dependant, thermosensitive polysaccharide hydrogels, aqueoussolutions used to form such hydrogels, and methods of preparation anduses thereof.

BACKGROUND OF THE INVENTION

Hydrogels are highly hydrated, macromolecular networks, dispersed inwater or other biological fluids.

Hydrogels that exhibit the specific property of increased viscosity withincreased temperatures are known as thermosensitive (or thermosetting)hydrogels. Such hydrogels have been shown to have easier applicationcombined with longer survival periods at the site of application ascompared to non-thermosensitive hydrogels, and are thereforeadvantageous as slow-release drug delivery systems.

It is known that thermosensitive hydrogels may be prepared from polymersof natural origin (O. Felt et al. in The Encyclopedia of Controlled DrugDelivery, 1999), such as chitosan, which is a commercially available,inexpensive polymer obtained by partial to substantial alkalineN-deacetylation of chitin, a linear polysaccharide, made ofN-acetylglucosamine units, linked via β-1,4 glycosidic bonds. Thedeacetylation process is generally performed using hot, concentrated,hydroxide solutions, usually sodium hydroxide.

Chitin is a naturally occurring biopolymer, found in the cytoskeletonand hard shells of marine organisms such as crustacea, shrimps, crabs,fungi, etc., and is the third most abundant naturally occurringpolysaccharide after cellulose and laminarine. Chitin is chemicallyinert, insoluble and, has a crystalline structure in the form of flakes,crumbs or tiles.

Chitosan contains free amine (—NH₂) groups and may be characterized bythe proportion of N-acetyl-D-glucosamine units to D-glucosamine units,commonly expressed as the degree of acetylation (DA) (reciprocal todeacetylation) of the chitin polymer. Both the degree of acetylation,and the molecular weight (MW) are important parameters of chitosan,influencing properties such as solubility, biodegradability andviscosity.

Chitosan is the only positively charged polysaccharide, making itbioadhesive, which delays the release of a medication agent from thesite of application (He et. al., 1998; Calvo et. al., 1997), and allowsionic salt interactions with anionic natural compounds such asglycoaminoglycans of the extra-cellular membrane.

Chitosan is biocompatible, non-toxic, and non-immunogenic, allowing itsuse in the medical, pharmaceutical, cosmetic and tissue constructionfields. For example, topical ocular applications and intraocularinjections or transplantation in the vicinity of the retina (Felt et.al., 1999; Patashnik et. al.; 1997; Song et. al., 2001). Moreover,chitosan is metabolized-cleaved by certain specific enzymes, e.g.lysozyme, and can therefore be considered as bioerodable andbiodegradable (Muzzarelli 1997, Koga 1998). In addition, it has beenreported that chitosan acts as a penetration enhancer by openingepithelial tight-junctions (Junginger and Verhoef, 1998; Kotze et. al.,1999), similar to the action of the enzyme hyaluronidase, the so called“spreading factor”. Chitosan also promotes wound-healing, as well asacting as an antiadhesive (preventing pathological adhesions) (Biaginiet. al., 1992; Ueno et. al., 2001) and exhibits anti-bacterial, (Feltet. al., 2000; Liu et. al., 2001), anti-fungal effects, and anti-tumorproperties.

Considering the remarkable properties of chitosan, there is a growingneed for new chitosan hydrogels for use in the growing industries ofslow-release of drugs and regenerative medicine.

Hydrogels comprising chitosan are very useful for drug delivery. Theymay conveniently be administered by local (intra-articular) routes; theyare injectable using minimally invasive procedures; drug delivery usinghydrogels provides a high level of concentration of the drug directly atthe target site; and they minimize adverse systemic effects and toxicityof the drug.

Chitosan microspheres have been developed for the delivery of drugs, inwhich drug release is controlled by particle size, degree of hydration,swelling ratio or biodegradability of the prepared microspheres.Attempts have been made to develop chitosan microspheres for thedelivery of drugs such as anti-cancer drugs, peptides, antibioticagents, steroids, etc. by cross-linking of chitosan to form a network.

Conventional chitosan cross-linking reactions have involved a reactionof chitosan with dialdehydes, which may have physiological toxicity.Novel chitosan networks with lower cytotoxicity were synthesized using anaturally occurring crosslinker called genipin, which providesbifunctional crosslinking by heterocyclic linking of genipin withchitosan by a nucleophilic attack and the formation of amide linkages(Mi et al., 2000).

The preparation of thermosensitive, neutral solutions based onchitosan/polyol salt combinations has been described by Chenite et al.,2000. These formulations possess a physiological pH and can be heldliquid below room temperature for encapsulating living cells andtherapeutic proteins; they form monolithic gels at body temperature,without any chemical modification or cross-linking. The addition ofpolyol salts bearing a single anionic head results in the formation of agel due to synergistic forces favorable to gel formation, such ashydrogen bonding, electrostatic interactions and hydrophobicinteractions. When injected in vivo the liquid formulation turns intogel implants in situ. The system has been used as a container-reservoirfor delivery of biologically active growth factors in vivo as well as anencapsulating matrix for living chondrocytes for tissue engineeringapplications.

Chitosan-glycerol phosphate/blood implants have been shown to improvehyaline cartilage repair in microfracture defects by increasing theamount of tissue and improving its biochemical composition and cellularorganization (Hoemann et al., 2005). The microfracture defect is filledwith a blood clot inhabited by bone-marrow derived cells that has beenstabilized by the incorporation of chitosan. The use of such implantswould therefore be expected to result in better integration, improvedbiochemical properties, and longer durability of the repair tissue.

Uniform submicron chitosan fibers, which may have an importantapplication as artificial muscles, as biosensors, or as artificial organcomponents, may be prepared by electro-wet-spinning technology (Lee etal. 2006).

Chitosan-based gels have been shown to turn into and serve as scaffoldsfor the encapsulation of invertebral disc (IVD) cells (Roughley et al.,2006), by entrapping large quantities of newly synthesized anionicprotcoglycan. Such gels would therefore be a suitable scaffold forcell-based supplementation to help restore the function of the nucleuspulposus structural region during the early stages of IVD degeneration.A denser, fibrillar collagen fabric may serve as an annulus fibrosisstructural substitute, allowing colonization with endogenous cells.

Collagen gel has previously been shown to be useful for repair ofarticular cartilage defects with cultured chondrocytes embedded in thegel (Katusbe et al., 2000). More recently, chitosan hydrogels have beenshown to be useful for cartilage regeneration and prevention of kneepain associated with acute and chronic cartilage defects.

Recently, temperature-controlled pH-dependant formation of ionicpolysaccharide gels, such as chitosan/organo-phosphate aqueous systems,has been described (WO 99/07416 and U.S. Pat. No. 6,344,488). Whilechitosan aqueous solutions are pH-dependent gelating systems, theaddition of a mono-phosphate dibasic salt of polyol or sugar to achitosan aqueous solution leads to further temperature-controlledpH-dependant gelation. Solid organo-phosphate salts are added anddissolved at low temperature within 0.5 to 4.0% w/v chitosan in aqueousacidic solutions. Aqueous chitosan/organo-phosphate solutions areinitially stored at low temperatures (4° C.), then endothermally gelatedwithin the temperature range of 30 to 60° C. Chitosan/organo-phosphatesolutions rapidly turn into gels at the desired gelation temperature.

An advanced clinical product of such chitosan hydrogels is a hydrogelproduced by BioSyntech. The thermosensitive chitosan hydrogel ofBioSyntech is prepared by neutralizing a commercial chitosan, having adegree of deacetylation of about 80-90%, with mono-phosphate dibasicsalts of polyols, particularly β-glycerophosphate (β-GP). Addition ofβ-GP to chitosan enables the pH to be increased up to about 7 withoutchitosan precipitation, and to form a hydrogel at that pH, atphysiological temperature.

A chitosan hydrogel (BST-CarGel™) is produced by BioSyntech, which fillscartilage defects and provides an optimal environment for cartilagerepair. The chitosan plasticizer mixture is delivered within a debridedcartilage defect following microfracture, using the patient's own bloodas a sole source of biological ingredients. The mixture fills the defectand solidifies in situ within 8-12 minutes, providing an effectivescaffold for cartilage regeneration. Healthy chondrocytes then migratefrom the deep inner bone through the microfracture pores and repopulatethe gel-filled lesion.

A second BioSyntech chitosan hydrogel, BST-DermOn™, may be used as atopical therapy for stimulating and supporting wound healing. Theproduct acts as a mucoadhesive barrier and can seal the wound andmaintain a moist environment while continuing to allow gas exchange.

A further BioSyntech chitosan hydrogel, BST-InPod™, is intended fortreatment of heel pain. This is an injectable product which is intendedto permanently restore comfort of plantar fat pads by integrating withthe patient's own pad fat and restoring biomechanical cushioningproperties and comfort.

These products, however, also exhibit some limitations. The BioSyntechproducts comprising commercially available chitosan, having a degree ofacetylation of about 15-20% DA, are believed to exert an undesiredslower degradation rate. Furthermore, chitosan has limited ability tomix with and encapsulate cells at physiological pH of 7.4 to form athree-dimensional scaffold.

The BioSynthech family of hydrogels has limited degradation rates andthe formation of such hydrogels requires the presence ofglycerophosphate or similar plasticizing salts. Glyerophosphate is anegatively charged molecular entity that can react with positive chargesof bioactive components, leading to their precipitation, or to thedisturbance of their release from the hydrogel. Therefore, the presenceof glycerophosphate may decrease the range of drugs with whichchitosan/glycerophosphate hydrogels can be used.

Further, the modulation of the properties of the hydrogel, such asgelation time and viscosity, depends on the concentration ofglycerophosphate, and is therefore limited by the solubility ofglycerophosphate. In particular, a high concentration ofglycerophosphate is required to have a low gelation time, avoiding therapid elimination of the hydrogel after its administration. However, ahigh concentration of glycerophosphate also decreases the viscosity ofthe hydrogel. Therefore, the gelation time has to be balanced with theconsistency of the hydrogel, and it is not possible to obtain hydrogelsthat have both low gelation time and high viscosity, which would be adesirable combination of characteristics. Also, a too high concentrationof glycerophosphate may induce the precipitation of the hydrogel at itsadministration site.

Further, thermosensitive chitosan/glycerophosphate gels were found to beturbid, thus rendering their use inappropriate for particularapplications such as ocular or topical administrations.

Multiple interactions are responsible for the solution/gel transition:the increase of chitosan interchain hydrogen bonding, as a consequenceof the reduction of electrostatic repulsion, due to the basic nature andaction of the salt, and the chitosan-chitosan hydrophobic interactionswhich should be enhanced by raising the pH. The gelation process thatoccurs upon increasing the temperature, predominantly originates due tothe strengthening of the chitosan hydrophobic attractions, also shown inthe presence of the glycerol moiety (serving as a plasticizer) andchitosan. At low temperatures, strong chitosan-water interactions,protects the chitosan chains from aggregation. Upon heating, sheaths ofwater molecules are removed, allowing the association of alignedchitosan macromolecules. Furthermore, electrostatic forces may decreaseupon raising the temperature, and the hydrophobic interactions areexpected to have a major contribution to the gelation of the chitosansmixture.

Transparent chitosan/glycerophosphate hydrogels have been prepared,requiring modification of deacetylation of chitosan by reacetylationwith acetic anhydride. The use of previously filtered chitosan, dilutionof acetic anhydride and reduction of temperature has been shown toimprove efficiency and reproducibility (Berger et al., 2004).

Turbidity of chitosan/glycerophosphate hydrogels has been shown to bemodulated by the degree of deacetylation of chitosan and by thehomogeneity of the medium during reacetylation, which influences thedistribution mode of the glucose amine monomers. The preparation oftransparent chitosan/glycerophosphate hydrogels therefore requires ahomogeneously reacetylated chitosan with a degree of deacetylationbetween 30 and 60%.

It has been found that reacetylation of commercial chitosan to producehomogenously acetylated chitosans having a degree of acetylation of fromabout 30% to about 60%, greatly increases the solubility of the chitosanin water and body fluids at physiological pH, without the need to useglycerol phosphate. Such chitosans produce clear transparent gels, whichmay be used for cell encapsulation (WO 05/097871 to Berger et al).

Homogeneous reacetylation of chitosan on one hand has the effect ofincreasing the number of hydrophobic sites by replacing amine groupswith acetyl groups, but on the other hand the crystalline structure thatmakes chitosan tend to fold is highly reduced cumulating in increasedsolubility of the chitosan. Reacetylation prevents refolding of thepolymer, maintaining the straight chain, and thus preventing thepH-related decrease in solubility.

An example of commercial chitosan which may be used in the preparationof reacetylated chitosan is a chitosan of pharmaceutical grade and highMW obtained from Aldrich Chemical, Milwaukee, USA, having a MW of 1100kDa as determined by size exclusion chromatographic method reported byO. Felt, et al. in Int. J. Pharm. 180, 185-193 (1999) and adeacetylation degree DD of 83.2% as measured by UV method reported by R.A. Muzarelli et al. in “Chitin in Nature and Technology”, Plenum Press,New York, 385-388, (1986).

However, any commercial chitosan having a deacetylation degree of 80 to90% and a molecular weight not smaller than 10 kDa may be used. Theacidic medium used for dissolving commercial chitosan may be for exampleacetic acid and the acidic solution of chitosan obtained aftersolubilization of chitosan may be then diluted with an alcohol, forexample methanol.

Generally, commercially available chitosan is industrially prepared bydeacetylation of dry chitin flakes (Muzzarelli, 1986). Deacetylationpreferentially occurs in the amorphous zones of the chitin molecules atthe surface of the flakes, resulting in non-homogeneous monomers withvariable block size of deacetylated-units distribution (Aiba, 1991). Incomparison, reacetylated chitosan under homogeneous conditions, adopts arandom distribution of deacetylated monomers, which induces a decreaseof the crystallinity of chitosan and in turn increases its solubility(Aiba, 1991, 1994; Ogawa and Yui, 1993; Milot et. al., 1998).

The suitability of polymeric hydrogels for an application is dictated bytheir biocompatibility, mechanical integrity, speed and reversibility ofgel formation at physiological pH, and their low weight and extendedlifetime. However, very little control is possible over variousimportant properties of known chitosan hydrogels, such as strength, rateof degradation, and release profile.

WO 10/109,460 to one of the inventors of the present inventors disclosesexpandable element comprising an injectable filler which compriseschitosans.

WO 03/011912 teaches a process of preparing chitosan in which in theheterogeneous deacetylation reaction of chitin, the latter is firstsubjected to a prolonged low temperature alkaline swelling stage. Theproduced chitosan thus may be obtained with a more random distributionof residual N-acetyl groups along the polymeric chains. The producedchitosan has a controllable degree of deacetylation, degree ofdepolymerization, and hence degree of water-solubility at physiologicalpH.

WO 2004/069230 teaches a pharmaceutical composition which comprises achitosan having an acetylation degree (F_(A)) of from 0.25 to 0.80(e.g., 0.3 to 0.6 or 0.33 to 0.55), which acts as a release sustainingor monoadhesive agent, and a physiological active agent. Someembodiments of WO 2004/069230 relate to a mixture of two or morechitosans having different acetylation degrees. Such mixtures preferablyinclude chitosans having an acetylation degree higher than 0.25, butsome are described as including one chitosan having an F_(A) value offrom 0.25 to 0.80 and one chitosan having an F_(A) value below 0.25, forexample, 0.05 to 0.19. The preparation of compositions having such amixture of chitosans, however, has not been described in thispublication.

The chitosans utilized in the compositions taught in WO 2004/069230 mayhave a weight average MW within a very broad range, and a very broadconcentration range. The taught compositions are powdered compositions,designed for oral administration.

WO 2004/068971 teaches foodstuffs comprising a nutritional foodsubstance and a chitosan having an acetylation degree (F_(A)) of from0.25 to 0.80, or a mixture of chitosans, as described in WO 2004/069230.The compositions and products taught in these publications are notdesigned so as to form a gel upon administration to a subject viainjection.

SUMMARY OF THE INVENTION

The present inventors have found that a composition comprising at leasttwo different types of chitosans, wherein the different types areclassified according to their degree of acetylation/deacetylation andoptionally according to the level of homogeneity of the acetylatedunits, provides a hydrogel in which a greater degree of control overvarious physical, chemical and pharmacokinetic properties is possible,particularly as compared to hydrogels comprising a single type ofchitosan.

The disclosed hydrogel composition is formed under physiologicalconditions, namely, physiological pH and temperature, and can thus beutilized in a myriad of applications, particularly medical applicationssuch as tissue regeneration, treatment of osteoarthritis, as alubricant, food additive, and the like.

The present invention provides a pH- and temperature-dependantcomposition for formation of a polysaccharide hydrogel at physiologicalconditions. According to one aspect, the present invention provides achitosan composition which forms a hydrogel at near physiological pH andat 37° C., comprising at least one acetylated chitosan having a degreeof acetylation in the range of from about 30% to about 60%, and at leastone deacetylated chitosan having a degree of deacetylation that is about70% to about 95%, the composition being in the form of an aqueoussolution at neutral pH, wherein the ratio of said acetylated chitosan tosaid deacetylated chitosan in the composition is in the range of 1:1 to4:1.

According to one embodiment, the chitosan composition further comprisingat least one negatively charged polysaccharide. According to anotherembodiment, the negatively charged polysaccharide is hyaluronic acid.

According to yet another embodiment, the chitosan composition furthercomprising acetylglucosamine oligomers. According to yet anotherembodiment, the acetylglucosamine oligomers compriseTri-N-acetyl-glucosamine. According to yet another embodiment, theacetylglucosamine oligomers are present in the composition at aconcentration of between 0.05% to 10% w/v.

According to yet another embodiment, the chitosan composition furthercomprising a cross-linking agent in an amount sufficient to accelerategelation of the chitosan composition. According to yet anotherembodiment, the cross-linking agent is capable of cross-linking theamine or hydroxyl residues of the chitosans. According to yet anotherembodiment, the amount of the cross linking agent is in the range ofbetween 0.1% to 2% w/v. According to yet another embodiment, thecross-linking agent is added to the chitosan composition prior tointroducing said chitosan composition into a site in vivo. According toyet another embodiment, the cross-linking agent is genipin.

According to another aspect, the present invention provides a processfor preparing a stable pH-dependent and temperature-dependent chitosanhydrogel composition comprised of at least one acetylated chitosanhaving a degree of acetylation of from about 30% to about 60%, and atleast one deacetylated chitosan having a degree of deacetylation that isabout 70% to about 95%, which process comprises:

a) dissolving said acetylated chitosan and said deacetylated chitosan inan acidic aqueous solution at a temperature between 0° C. to 10° C., tothereby form a composite solution, wherein the ratio of said acetylatedchitosan and said deacetylated chitosan in said composite solution is inthe range of 1:1 to 4:1;

b) adjusting the pH of said composite solution to a value of 6.6 to 7.4at a temperature between 0° C. to 10° C.; and

c) increasing at least one of the temperature of said composite solutionto about 37° C. and/or raising the pH to physiological pH.

According to one embodiment, the process further comprising adding anegatively charged polysaccharide to the composition before step c).According to another embodiment, the polysaccharide is hyaluronic acid.

According to yet another embodiment, the process further comprisingadding acetylglucosamine oligomers to the composition.

According to yet another embodiment, the process further comprisingadding a cross-linking agent in an amount sufficient to accelerategelation of the chitosan composition, wherein the cross-linking agent isadded to the composition prior to introducing said chitosan compositioninto a site in vivo. According to yet another embodiment, thecross-linking agent is genipin.

According to yet another aspect, the present invention provides ahydrogel chitosan composition formed by the process of the invention.

According to yet another aspect, the present invention provides a methodof treating medical condition in a subject in need thereof, comprisingapplying into a site of the medical condition a chitosan compositioncomprising at least one acetylated chitosan having a degree ofacetylation in the range of from about 30% to about 60%, and at leastone deacetylated chitosan having a degree of deacetylation that is about70% to about 95%, the composition being in the form of an aqueoussolution at neutral pH, wherein the ratio of said acetylated chitosan tosaid deacetylated chitosan in the solution is in the range of 1:1 to4:1.

According to one embodiment, the medical condition is selected from thegroup consisting of: osteoarthritis, fracture repair, bone structuralsupport, cartilage repair, intervertebral disc repair, meniscal repair,bone reconstruction, bone filling and synovial fluid replacement.

According to yet another aspect, the present invention provides a kitfor producing a chitosan hybrid hydrogel, comprising:

(i) a container containing a chitosan composition comprising at leastone acetylated chitosan having a degree of acetylation in the range offrom about 30% to about 60%, and at least one deacetylated chitosanhaving a degree of deacetylation that is about 70% to about 95%, whereinthe ratio of said acetylated chitosan to said deacetylated chitosan inthe solution is in the range of 1:1 to 4:1;

(ii) a container containing a cross-linking agent in an amountsufficient to accelerate gelation of the chitosans; and

(iii) instructions for preparing the chitosan hybrid hydrogel.

According to one embodiment, the container containing a chitosancomposition further comprises a negatively charged polysaccharide.According to another embodiment, the polysaccharide is hyaluronic acid.

According to yet another embodiment, the container containing a chitosancomposition further comprises acetylglucosamine oligomers. According toyet another embodiment, the acetyl glucosamine oligomers compriseTri-N-acetyl-glucosamine. According to yet another embodiment, the kitfurther comprises a container containing a solvent for dissolving thecross-linking agent.

According to another aspect of the present invention there is provided amethod for the production of a stable hydrogel which comprises acomposition of at least one highly acetylated chitosan having a degreeof acetylation of from about 30 to about 60%, and at least one highlydeacetylated chitosan having a degree of deacetylation of from about70%. The method is effected by dissolving at least one highly acetylatedchitosan and at least one highly deacetylated chitosan in an acidicaqueous solution, to form a composite solution; adjusting the pH of thecomposite solution to a value of from 6.5 to 7.2; and increasing thetemperature of the composite solution to 37° C. while raising the pH tofrom 7.0 to 7.6.

The chitosan gel resulting from this mixture of at least two chitosantypes may optionally comprise microspheres of chitosan encapsulating adrug and/or electrospun chitosan fibers embedded in the gel.

In some embodiments, the highly acetylated chitosan is eitherhomogenously acetylated or homogenously deacetylated. In someembodiments, the highly deacetylated chitosan is non-homogenouslydeacetylated.

The highly acetylated chitosan and the highly deacetylated chitosan mayoptionally each be present at a concentration of from about 0.1% toabout 6% w/v of the total composition, and may each have a molecularweight in the range of from about 10 kDa to about 4000 kDa. In someembodiments, the highly acetylated chitosan and the highly deacetylatedchitosan are each present at a concentration of from about 0.2% to about3% w/v of the total composition. Alternatively, the highly acetylatedchitosan and the highly deacetylated chitosan are each present at aconcentration of from about 0.5% to about 2% w/v of the totalcomposition. In some embodiments, the highly acetylated chitosan and thehighly deacetylated chitosan are each present at a concentration of fromabout 1% to about 1.2% w/v of the total composition.

In some embodiments, the highly acetylated chitosan has a molecularweight of greater than about 200 kDa. In some embodiments, the highlydeacetylated chitosan has a molecular weight of greater than about 60kDa. Alternatively, the highly deacetylated chitosan has a molecularweight of greater than 200 kDa. Further alternatively, the highlydeacetylated chitosan has a molecular weight of greater than about 400kDa.

Further alternatively, the highly deacetylated chitosan has a molecularweight that ranges from 400 kDa to 700 kDa and the highly acetylatedchitosan has a molecular weight that ranges from 200 kDa to 250 kDa.Further alternatively, in this embodiment, each of the highly acetylatedchitosan and the highly deacetylated chitosan is present at aconcentration of from 1% to 1.2% (w/v) of the composition. Furtheralternatively, the ratio of the highly acetylated chitosan and thehighly deacetylated chitosan is 1:1.

The properties of the hydrogel may be controlled by manipulation of themolecular weight, degree of deacetylation and distribution of thedeacetylated sites of both the highly acetylated chitosan and the highlydeacetylated chitosan. These manipulations will influence the gelproperties, such as, for example, the gelation temperature, density orporosity, or the degree of hydration or the degree of hydrophobicity.Alternatively, properties of the hydrogel such as gelation temperatureand gelation pH can be controlled by manipulation of the molecularweight, the concentration and ratio of the chitosan types.

Further alternatively, the manipulation comprises manipulation of afeature selected from the group consisting of a molecular weight of eachof the highly acetylated chitosan and the highly deacetylated chitosan,a concentration of each of the highly acetylated chitosan and the highlydeacetylated chitosan in the composition and a ratio of the highlyacetylated chitosan and the highly deacetylated chitosan.

In one embodiment, when a concentration of each of the highly acetylatedchitosan and the highly deacetylated chitosan ranges from 1% to 1.2% w/vof the composition and the ratio is 1:1, the hydrogel is formed at nearphysiological pH. In one embodiment, the highly deacetylated chitosanhas a molecular weight of from 400 kDa to 700 kDa and the highlyacetylated chitosan has a molecular weight of from 200 kDa to 250 kDa.In one embodiment, when the highly deacetylated chitosan has a molecularweight of at least 2000 kDa and a concentration thereof is 0.5%, andfurther when the highly acetylated chitosan has a molecular weight offrom 200 kDa to 250 kDa and the ratio ranges from 2:1 to 4:1, thehydrogel is formed at the near physiological pH.

The degradation rate of the hydrogel may be further controlled bybinding of the lysozyme inhibitor Tri-N-acetyl-glucosamine to the highlyacetylated chitosan.

The chitosan composition may optionally further comprise a negativelycharged polysaccharide, such as, for example, an animal- orplant-derived polymer. As a non-limiting example of a plant-derivedpolymer, the negatively charged polysaccharide may optionally compriseseaweed. Alternatively, the negatively charged polysaccharide mayoptionally comprise a glycosaminoglycan, such as, for example,hyaluronic acid, chondroitin sulfate, or other acidic polymers such asdextran sulfate. According to one embodiment, the chitosan compositioncomprises hyaluronic acid. Alternatively, the chitosan composition maycomprise other negatively charged substances, such as, for example,phospholipids. A non-limiting example of a phospholipid is phosphatidylcholine.

Further alternatively, the chitosan composition comprises both aglycosaminoglycan and a phospholipid, as described herein. Such acomposition is suitable for use as a synovial replacement composition.According to some embodiments, the chitosan composition described hereinmay further comprise at least one of a drug, a polypeptide and a cell(such as an animal cell or a plant cell).

The composition may further comprise an emulsifier. Optionally, thechitosans and the emulsifier may form nanoparticles. Optionally, thenanoparticles are encapsulated in the hydrogel.

The chitosan composition described herein can be used to form a hydrogelwhich is beneficially utilized in an application such as, for example,drug delivery, food additive delivery (via oral ingestion), support ofcell growth, bone structural support, cartilage repair, tissuereconstruction, wound-healing, production of artificial skin, as ahypolipidemic and hypocholesterolemic agent (for treating hypolipidemiaand hypocholesterlimia), formation of artificial kidney membrane, bonefilling, soft tissue reconstruction as for heel pain relief for example,anti adhesion in the field of surgery, lubrication, synovialreplacement, apophysitis, tenditinitis, mesotherapy, burn treatment, andinflammatory arthritis. The chitosan composition which forms thehydrogel can be administered by a route such as injection or endoscopicadministration.

The formed hydrogel may optionally be used in the preparation of abiocompatible material for use in the preparation of an implantabledevice, such as for use in tissue repair, tissue reconstruction, tissueconstruction, and tissue replacement.

In some embodiments, the anti-adhesion properties of chitosan makes thisgel useful as an anti adhesion device in applications such as cardiothoracic surgery and abdominal surgery, for example.

According to some embodiments, the formed hydrogel may optionally beused in the preparation of a drug delivery device or system. The drugdelivery device or system may optionally provide slow release of anembedded medication. Non-limiting examples of drugs for use in thissystem include proteins (such as BSA or hemoglobin) or non-proteinagents (such as, for example, ACE-inhibitors or anti inflammatorydrugs). The drug delivery system may optionally be an opthalmologicaldrug delivery system due to the transparency of the gel. However thedrug delivery system may also optionally be implemented for urologicalapplications such as vesicoureteral reflux and in cosmetic applicationssuch as for example wrinkle treatment.

The drug may also optionally comprise one or more of a mineral, avitamin, a food additive or natural extract such as a plant derivedextract for example. The gel itself, optionally with an activeingredient, may optionally be used as a food additive. In someembodiments, the drug comprises autologous cells, and the system isbeing for delivering the cells into rotator-cuff tears and/or tendondamage. Such delivering can be performed under ultrasonographic control.

Alternatively, the formed hydrogel may optionally be used for supportingendogenous cells in a three-dimensional gel construct. As a furtheralternative, the formed hydrogel may optionally be used for embeddingexogenous cells with or without added growth factors, as well the cellmay provide metabolites such as growth factors. Also alternatively, thehydrogel may optionally be used in the production of a cell-loadedartificial matrix, where the cells are, for example, chondrocytes,fibrochondrocytes, ligament fibroblasts, skin fibroblasts, tenocytes,myofibroblasts, mesenchymal stem cells and keratinocytes.

According to some embodiments, there is provided a chitosan compositioncomprising nanoparticles containing an active ingredient andencapsulated in a hydrogel comprising at least one type of chitosanhaving a degree of acetylation in the range of from about 30% to about60%, and at least one type of chitosan having a degree of deacetylationof at least about 70%, wherein the hydrogel is formed through pH- andtemperature-dependant gelation. Optionally, the composition furthercomprises an emulsifier. Also optionally, the hydrogel forms uponinjection to a subject.

According to some embodiments, the chitosan hydrogel may optionally beused as a lubricating agent in such applications such as vaginalatrophy, dry eyes, conjunctivitis sicca, dry nose following upperrespiratory infections as well as a general soothing agent for variousabrasions. Chitosan gel may also optionally be used as ananti-inflammatory agent in facial diseases such as fibromyalgia byeither local injection or external massage.

According to some embodiments, the chitosan hydrogel is used in a methodof mesotherapy which is effected by injecting the chitosan compositiondescribed herein. Alternatively, the chitosan hydrogel formed from thechitosan composition described herein can be used as a synovialreplacement composition, for use in the treatment of, for example,osteoarthritis. Such a synovial replacement composition is describedhereinabove.

According to an aspect of some embodiments of the invention there isprovided a process of preparing a stable hydrogel which comprises ahighly deacetylated chitosan and a highly acetylated chitosan, asdescribed herein. Further, a hydrogel prepared by this process isprovided.

According to a further aspect of some embodiments of the presentinvention there is provided a chitosan composition which forms ahydrogel at near physiological pH and 37° C., the composition comprisingat least one type of a highly deacetylated chitosan having a molecularweight of from 10-4000 KDa and a degree of deacetylation of at least70%, and at least one type of a chitosan having a molecular weight offrom 200-20000 Da, the composition being in a form of an aqueoussolution.

In some embodiments, the chitosan having a molecular weight of from200-20000 Da is selected from the group consisting of a highlydeacetylated chitosan having a degree of deacetylation of at least 70%and a highly acetylated chitosan having a degree of acetylation of from30% to 60%.

In some embodiments, a ratio of the chitosan having a molecular weightof from 200-20000 Da and the highly deacetylated chitosan having amolecular weight of from 10-4000 KDa and a degree of deacetylation of atleast 70% is higher than 1:1. In some embodiments, the ratio ranges from2:1 to 20:1.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 illustrates the formation of a hydrogel according to someembodiments of the present invention from a liquid compositioncomprising two different types of chitosan;

FIG. 2 a illustrates degradation times of a hydrogel compositioncomprising chitosan type 1 and type 2 in accordance with someembodiments of the present invention;

FIG. 2 b illustrates degradation times of a hydrogel compositioncomprising chitosan type 1 and type 2 at different ratios;

FIG. 3 illustrates release of hemoglobin from a hydrogel compositionaccording to some embodiments of the present invention, as measured byμg/ml in eluent;

FIG. 4 illustrates release of bovine serum albumin (BSA) from a hydrogelcomposition according to some embodiments of the present invention asmeasured by optical density (OD);

FIG. 5 presents a bar chart illustrating release of BSA from a hydrogelcomposition according to some embodiments of the present invention;

FIG. 6 illustrates the degradation profile of a hydrogel compositionaccording to some embodiments of the present invention;

FIG. 7 illustrates the integration of the release profile of BSA withthe degradation profile of a hydrogel composition according to someembodiments of the present invention;

FIGS. 8A and 8B show histopathology of wound bed biopsies taken fromrats treated with a hydrogel composition according to some embodimentsof the present invention;

FIG. 9 shows a graph of the results of treating wound bed biopsies witha hydrogel composition according to some embodiments of the presentinvention;

FIGS. 10A and 10B show the results of in vivo experiments performed onrats for rotator cuff damage;

FIG. 11 presents a schematic illustration of measuring a frictioncoefficient; and

FIG. 12 show the static friction coefficient of hydrogel compositionsaccording to some embodiments of the present invention, compared to ahyaluronic acid composition.

FIG. 13 shows histology micrographs presenting rat knees followingmedial meniscectomy which were further treated with a compositioncomprising chitosans and hyaluronate (panel a) or with control (panelb).

FIG. 14 shows micrographs of environmental scanning electron microscopyof a composition comprising chitosans and hyaluronate (panel a) or acomposition comprising chitosans only (panel b).

FIG. 15 presents micrographs showing that subcutaneous injection in ratsof a chitosan composition comprising hyaluronate does not evoke aninflammatory response. The gel forms a discrete nodule (arrow); originalmagnification ×10.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates topositively charged polysaccharide hydrogels, and, more particularly, topH-dependent, thermosensitive polysaccharide hydrogels formed from anaqueous solution of polysaccharides, to such solutions ofpolysaccharides and to methods of preparation and uses of pH-dependent,thermosensitive polysaccharide hydrogels.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Chitosans which are deacetylated to a degree of deacetylation (alsoreferred to herein as DD or DDA) of about 70-100% (i.e. degree ofacetylation, DA, of up to about 30%), such as commercially availablechitosan, are termed herein Type 1 chitosans or chitosans type 1. Thesechitosans are insoluble at physiological pH, and are poorly recognizedby lysozyme. Such chitosans, when utilized in in vivo applications, aretypically characterized by relatively slow biodegradation, which,depending on the degree of deacetylation, can last from a few days to afew months. Gels formed by chitosans of this type have a low degree ofacetylation, such that the free amine groups participate in densehydrogen bonds with many hydrophobic interactions.

The degradation rate of chitosans has been shown to be a function of thedegree of deacetylation. Degradation of chitosan has an influence oncell proliferation and remodeling.

Highly homogeneously deacetylated or reacetylated chitosans (havingdegree of acetylation of from about 30% to about 60%) are termed hereintype 2 chitosans or chitosans type 2. Such chitosans are readilydigested/degraded by lysozyme, thereby enabling, for example, controlleddrug release of a drug encapsulated therein.

If deacetylation degree of chitosan is lower than 30%, the chitosanbecomes a polymer close to chitin that is insoluble in acidic conditionsand therefore not suitable for use in embodiments of the presentinvention. At a degree of deacetylation greater than 70%, precipitationof chitosan occurs.

The deacetylation degree of chitosan may be determined by aspectrophotometric method such as described, for example, in theliterature by R. A. Muzarelli and R. Richetti [Carbohydr. Polym. 5,461-472, 1985 or R. A. Muzarelli and R. Richetti in “Chitin in Natureand Technology”, Plenum Press 385-388, 1986]. Briefly, in the lattermethod for example, chitosan is solubilized in 1% acetic acid and the DDis determined by measuring its content of N-acetyl-glucosamine by UV at200, 201, 202, 203 and 204 nm using N-acetyl-D-glucosamine solutions asstandards.

According to one embodiment, the present invention relates to apolysaccharide (chitosan) composition comprising a combination of atleast one highly acetylated chitosan (type 2) having a degree ofacetylation of from about 30% to about 60%, and at least one highlydeacetylated chitosan (type 1), having a degree of deacetylation of atleast 70%.

The highly acetylated type 2 chitosans can interact throughelectrostatic, hydrogen and hydrophobic interactions with the highlydeacetylated chitosans type 1. The extent of interaction increases withincreasing pH. A composition comprising solutions of both types ofchitosan can form a stable gel at physiological pH, without the need forglycerophosphate. The obtained composition is therefore devoid ofglycerophosphate.

Thus, the chitosan composition described herein is in a form of anaqueous solution, and forms a hydrogel at physiological conditions(e.g., near physiological pH and 37° C.).

It is noted that the composition described herein can form a gel also atroom temperature or at lower temperatures (e.g., 4° C.). Nonetheless,the gel formation at such conditions is slow and may last from a fewdays to a few months, thus enabling to store and transfer thecomposition as an aqueous solution.

Type 1 chitosans precipitate at a pH of about 6.5, which is less thanphysiological pH. Interaction of the highly hydrophobic, homogenous,chitosan type 2 with chitosan type 1 prevents this precipitation of thenon-homogenously acetylated type 1 chitosan, by formation of hydrogenand hydrophobic bonds, allowing the formation of a stable solution at pHof about 6.7, and a stable semi-solid hydrogel at about pH 7.0 andabove.

The secondary bonds which are formed allow the encapsulation of thenon-homogenous chitosan chains and maintain its solubility at pH greaterthan its pKa value. Generally, such secondary chain interactions are themain molecular forces involved in gel formation (Chenite et. al., 2000;Berger et. al., 2005).

Type 1 chitosans mainly contribute to the stability, strength andrigidity of the gel, and provide slow degradation, while type 2chitosans contribute to the softness, elasticity and fast solubilizationof the gel. The different degradation profiles of type 1 and type 2chitosans are discussed further in Example 2 below, and are shown inFIG. 2. The type 2 chitosan can be regarded as a “protector” or“coating”, which provides a shell around the type 1 chitosan and thusavoids its precipitation and delays its biodegradation.

Furthermore, the type 2 chitosan is recognized by lysozyme. This featureenables to control the degradation rate of a hydrogel formed from thecomposition described herein. For example, binding a lysozyme inhibitorto the type 2 chitosan can slow the degradation rate of the formedhydrogel. Alternatively, the type 2 chitosan coating is recognized bythe lysozyme and is subjected to faster degradation. See, for example,Example 4 hereinbelow.

According to one embodiment, the chitosan composition of the inventionmay comprise a cross-linking agent. The cross-linking agent is providedin an amount sufficient to accelerate gelation of the chitosans.According to some embodiments, a sufficient amount of the cross linkeris an amount in the range of between 0.1% to 2% w/v, 0.1% to 1.8%, 0.1%to 1.6%, 0.1% to 1.4%, 0.1% to 1.2%, 0.1% to 1%, 0.1% to 0.8%, 0.1% to0.6%, or 0.1% to 0.4%. According to some embodiments, even lower amountsof the cross linking are sufficient to accelerate gelation. Inaccordance with those embodiments, the cross linker amount is in therange of between 0.01% to 0.1%, 0.01% to 0.05% or 0.05% to 0.1%.

The term “accelerated gelation” is used to denote that gelation occurssufficiently rapidly to prevent leakage of the gel formulation afteradministration of the chitosan mixture composition in vivo. Mostnotably, rapid gelation is critical for treating intervertebral discdefects to prevent leakage upon removal of the device used foradministration. In some cases, a desirable accelerated gelation is onethat causes the composition to completely gel in the time between whenthe cross-linking agent is added and the time that the composition isadministered. In some cases, gelation occurs almost instantaneouslyafter the addition of the cross-linking agent. The gelation time canalso depend upon the amount of cross-linking agent that is added and askilled artisan can determine the preferred amounts and types ofcross-linking agents for any particular chitosan composition based onroutine testing.

The addition of a cross linking agent to the chitosan composition of theinvention is also advantageous in that it reduces the inflammatoryreaction that may be associated with chitosans injection in vivo. Inaddition, the cross-linker accelerates gelation of the chitosans andthus avoids linkage of the chitosan composition following injection invivo.

According to some embodiments, the cross linking agent is added andadmixed with the chitosan composition adjacent or prior to injection ofthe composition of the invention into a site in vivo. According to oneembodiment, a site in vivo is a site of a medical condition. Accordingto some embodiments, adjacent or prior to is no more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25 or 30 minutes. Each possibility represents aseparate embodiment of the invention.

According to some embodiments, the cross linker is selected from thegroup consisting of: divinylsulfon, genipin and glutaraldehyde. Eachpossibility represents a separate embodiment of the invention. Accordingto one embodiment, the cross-linker is genipin.

According to some embodiments, the chitosan composition furthercomprises acetylglucosamine oligomers. According to some embodiments,the chitosan composition further comprises N-acetylglucosamine orOligo-N-acetyl-glucosamine. According to some embodiments, theN-acetylglucosamine may comprise 1-7 monomeric units. Theacetylglucosamine oligomers may be present in the composition at aconcentration of from 0.05% to 10% w/v, from 0.05% to 9%, from 0.05% to8%, from 0.05% to 7%, from 0.05% to 6%, from 0.05% to 5%, from 0.05% to4%, from 0.05% to 3%, from 0.05% to 2%, from 0.05% to 1%, or from 0.05%to 0.5%.

The physical and chemical properties of the hydrogel formed from thedescribed composition are altered by raising or lowering the molecularweight of the chitosans and/or their degree of acetylation, and by thenatural acetylation diversity of chitosans from different sources. Theproperties of the gel can further be controlled by selection of the typeof reacetylation (i.e. homogenous or non-homogenous), or by mapping thepatterns of distribution of the deacetylated/acetylated sites.

A hydrogel should be regarded as a viscous or semi-solid jelly-likemacromolecular network structure that swells in water. Themacromolecular network is made up of hydrophilic polymer units that areheld together either solely by non-covalent bonds or additionally by acertain amount of covalent bonds. The non-covalent hydrogels, betterknown as uncross-linked networks, can be soluble in water. The covalentnetworks, better known as cross-linked hydrogels, are water-insoluble.Crosslinked hydrogels can be prepared from uncrosslinked hydrogels viaan intra- or intermolecular chemical reaction of mutually reactivefunctional groups. If necessary cross-linking can be accomplished via across-linking agent as described hereinabove.

Preferably, the highly acetylated chitosan is homogenously acetylated.Further preferably, the highly deacetylated chitosan is non-homogenouslydeacetylated.

Increasing the molecular weight of the chitosan increases its viscosity,such that the polymer is highly hydrated and highly hydrophobic. A gelformed from such a polymer therefore has an increased strength, andgreater water retention. This results in a slow degradation rate, slowdrug release, and improved mechanical properties.

Preferably, each of the highly acetylated and highly deacetylatedchitosans has a molecular weight of from about 10 kDa to about 4000 kDa.Molecular weight of chitosan may be easily determined by size exclusionchromatography as reported for example by O. Felt, P. Purrer, J. M.Mayer, B. Plazonnet, P. Burri and R. Gurny in Int. J. Pharm. 180,185-193 (1999). The upper limit of MW is determined by the required easeof administration, which depends on the chosen application.

Increasing the degree of acetylation results in increased hydrophobicityin the range of 0-30% DA, but at higher values, such as 30-60% DA, thepolymer begins to become more and more soluble as the amount of DA isincreased. Furthermore, increasing the number of acetyl glucosaminegroups increases the rate of degradation in the body, due to increasedrecognition sites for lysozyme. Hence, the rate of release of thehydrogel from the body can be controlled by varying the degree ofchitosan acetylation.

Variations in the molecular weight, degree of deacetylation and thedistribution of the acetylated sites, concentration and ratio of the twoor more chitosans, affect the conditions (pH, temperature etc.) underwhich gel formation occurs; solubility; biodegradability; degree ofreactivity with proteins, active pharmaceutical ingredients or otherchemicals; hydrophobicity/hydrophilicity; degree of hydration; as wellas biological and biocompatibility properties of the gel, such as effecton cell growth, proliferation and survival, ability of chitosans tofunction as inflammatory or anti-inflammatory mediators, and the effectof chitosans on acceleration or deceleration of wound healing.

For example, Type 1 chitosans of higher molecular weight have higherhydrophobicity and higher viscosity, resulting in a stronger gel due tohigher inter-molecular interactions. Type 1 chitosans of higher DDA havea lower rate of degradation. Type 1 chitosans having highercrystallinity have a lower degradation rate due to the fact that thecrystalline form is non-soluble. Hence one skilled in the art canpredict properties of the resultant gel mixture, and would therefore beable to create gels having desired characteristics, using uniquecombinations of the different types of chitosan.

As shown in the Examples section that follows (see, Example 7),variation of the molecular weight of the chitosans used, theconcentration thereof and the ratio between the type 1 chitosan and type2 chitosan affect the conditions at which gel formation occurs. Hence,one skilled in the art can be able to select the appropriate parametersin order to produce a hydrogel under physiological conditions.

Preferably, each of the highly acetylated and the highly deacetylatedchitosans is independently present at a concentration of about 0.1% to6% w/v of the total composition.

In some embodiments, each of the highly acetylated and the highlydeacetylated chitosans is independently present at a concentration ofabout 0.2% to 3% w/v of the total composition.

In some embodiments, each of highly acetylated and the highlydeacetylated chitosans is independently present at a concentration ofabout 0.5% to 2% w/v of the total composition.

In some embodiments, each of highly acetylated and the highlydeacetylated chitosans is independently present at a concentration ofabout 1% to 1.2% w/v of the total composition.

In some embodiments, the ratio between the highly acetylated and thehighly deacetylated chitosans is 1:1, such that the above concentrationsare for each of the highly acetylated and the highly deacetylatedchitosans.

In other embodiments, and depending, for example, on the molecularweight of the chitosans used, as well as their concentration, the ratiocan be 2:1, 3:1 and even 4:1. Also contemplated are ratios such as1.1:1, 1.2:1, 1.5:1 1.8:1, and any other ratio in the range of from 1:1to 4:1. The ratios of the chitosans are ratios of w/v %.

In general, it can be assumed that increasing the MW of any of thechitosans used for forming the hydrogel, and particularly the type 1chitosan, allows decreasing its concentration and vise versa, decreasingthe MW of the chitosan requires increased concentration thereof, inorder to form a hydrogel.

The composition described herein offers greater possibility ofcontrolling the properties of the formed hydrogel, including, forexample, the hydrogel strength, rate of degradation, and release rate,as compared to the Chitosan/βGP based hydrogel patented by BioSyntech,and extends the possibilities of controlling the gel's properties, andtailoring them to the needs of a much wider range of chemical andphysical uses.

The hydrogel of the present invention may further comprise a thirdchitosan, selected from either type 1 or type 2, having a differentmolecular weight or degree of deacetylation, thus extending control overthe resultant hydrogel.

The polysaccharide hydrogel according to the present invention mayoptionally comprise a hybrid of chitosan with a negatively chargedsubstance. Such a substance can be, for example, a negatively chargedpolysaccharide, such as a glycosaminoglycan, for example, hyaluronicacid or chondroitin sulfate. It should be understood that hyaluronicacid is interchangeable with hyaluronate.

Such a substance can also be a phospholipid. A hybrid with aphospholipid is highly beneficial for use as a synovial replacement inosteoarthritis treatment, as it lowers the friction between cartilagesurfaces (see, Example 6 below).

In some embodiments, the chitosan composition described herein furthercomprises both a glycosaminoglycan and a phospholipid.

Thus, different compositions and mixtures based on these two types ofchitosans may be used to provide semi-solid hydrogels with suitableproperties for a wide range of applications. Exemplary applicationsinclude, but are not limited to drug delivery systems e.g. for slowrelease of agents or medications, scaffolding of various consistencies,including gels for supporting cell growth or bone structural support;cartilage repair; tissue reconstruction; in wound-dressings, promotingscar free healing and macrophage activation; for production ofartificial skin; as a hypolipidemic and hypocholesterolemic agent; as anartificial kidney membrane; for bone filling; and heel pain relief,arthritis, arthrosis, tendinitis, tendinosis, apohysitis, myositis,myalgia, taut muscular bands, soft tissue inflammation and as synovialreplacement composition for, for example, treating osteoarthritis.

The hydrogel may be formed in situ (in vivo) sub-cutaneously,intra-peritoneally, intra-muscularly or within biological connectivetissues, bone defects, fractures, articular cavities, body conduits orcavities, eye cul-de-sac, or solid tumors.

The polysaccharide solution may be introduced within an animal or humanbody by injection or endoscopic administration.

Drugs, polypeptides, living microorganisms, animal or human cells may beincorporated within the polysaccharide solution prior to gelation.

In accordance with the present invention there is also provided the useof the polysaccharide gel formed from the compositions described hereinfor producing biocompatible degradable materials used in cosmetics,pharmacology, medicine and/or surgery.

Herein, a hydrogel is referred to a semi-solid gel formed from thechitosan aqueous solutions described herein, upon subjecting thesesolutions to the physiological conditions described herein. The hydrogelis preferably formed in vivo, upon administration of the chitosancomposition, but can alternatively be formed ex-vivo prior to itsutilization.

The gel may be incorporated as a whole, or as a component, intoimplantable devices or implants for repair, reconstruction and/orreplacement of tissues and/or organs, either in animals or humans.

The gel may be used as a whole, or as a component of, implantable,transdermal or dermatological drug delivery systems.

The gel may be used as a whole, or as a component of, opthalmologicalimplants or drug delivery systems.

The gel may be used for producing cells-loaded artificial matrices thatare applied to the engineering and culture of bioengineered hybridmaterials and tissue equivalents.

The loaded cells may be selected from the group consisting ofchondrocytes (articular cartilage), fibrochondrocytes (meniscus),ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes(tendons), myofibroblasts (muscle), mesenchymal stem cells,keratinocytes (skin), and neurons, as well as adipocytes or bone marrowcells. In fact cells from any tissue which are capable of proliferationmay optionally be embedded in such a construct.

A major detriment to wound heeling is the presence of biofilm. Biofilmis composed of at least 80 percent extracellular macromolecules that areusually positively charged, similar to chitosan. Chitosan may optionallybe used as a biofilm disruptor thus helping wound hygiene and limitingthe inhibitory effect of biofilm on destruction of bacteria. Chitosangel mixed with lactoferrin may optionally act as a slow releasereservoir to destroy biofilm in any chronic wound or a wound that maybecome chronic. Chitosan gel mixed with xylitol may optionally also be aspecific biofilm disruptor.

In accordance with the present invention there is also provided the useof loaded polysaccharide gel as injectable or implantable gelbiomaterials which act as supports, carriers, reconstructive devices orsubstitutes for the formation in situ of bone-like, fibrocartilage-likeor cartilage-like tissues at a physiological location of an animal or ahuman.

For example, chitosan gels according to the present invention may beuseful as a sustained delivery drug-system for treatment of the eye.Results based on the ocular irritation test of chitosan compounds haveindicated that chitosan preparations are suitable for use as ophthalmicgels based on their excellent tolerance (Molinaro et. al., 2002).

In accordance with a further embodiment of the present invention, a slowrelease drug delivery hydrogel system is provided comprising highlyacetylated type 1 chitosans and highly deacetylated type 2 chitosans.

Any of the drug delivery systems of the present invention may be usedfor delivery of a wide variety of drugs, including, but not limited to,analgesics, anesthetics, antiacne agents, antiaging agents,antibacterials, antibiotics, antiburn agents, antidepressants,antidermatitis agents, antiedemics, antihistamines, antihelminths,antihyperkeratolyte agents, antiinflammatory agents, antiirritants,antilipemics, antimicrobials, antimycotics, antioxidants, antipruritics,antipsoriatic agents, antirosacea agents antiseborrheic agents,antiseptics, antiswelling agents, antiviral agents, antiyeast agents,cardiovascular agents, chemotherapeutic agents, corticosteroids,fungicides, hormones, hydroxyacids, keratolytic agents, lactams,mitocides, non-steroidal anti-inflammatory agents, pediculicides,progestins, sanatives, scabicides, and vasodilators.

In some embodiments, the drug is an ACE inhibitor or ananti-inflammatory drug.

In accordance with a further embodiment of the present invention, Aprocess for preparing a stable pH-dependent and temperature-dependentchitosan hydrogel composition comprised of at least one acetylatedchitosan having a degree of acetylation of from about 30% to about 60%,and at least one deacetylated chitosan having a degree of deacetylationthat is about 70% to about 95%, which process comprises:

a) dissolving said acetylated chitosan and said deacetylated chitosan inan acidic aqueous solution at a temperature between 0° C. to 10° C., tothereby form a composite solution, wherein the ratio of said acetylatedchitosan and said deacetylated chitosan in said composite solution is inthe range of 1:1 to 4:1;

b) adjusting the pH of said composite solution to a value of 6.6 to 7.4at a temperature between 0° C. to 10° C.; and

c) increasing at least one of the temperature of said composite solutionto about 37° C. and/or raising the pH to physiological pH.

It is to be noted that the composite solution may present different pHvalues at different temperatures. In example, pH values at 4° C.correlate to pH values which are lower by about 0.5 units at 25° C.Thus, a composite solution at 4° C. with pH 7.4 may present about pH 6.9at 25° C.

In some embodiments, dissolving of the highly acetylated chitosan andthe highly deacetylated chitosan is performed simultaneously in the samevessel.

Optionally, dissolving the highly acetylated chitosan and the highlydeacetylated chitosan is performed in separate vessels to form twosolutions. In such embodiment, the process further comprises mixingthese two solutions to form the composite solution.

Further according to some embodiments of the present invention there isprovided a pH-dependant and temperature-dependant hydrogel formed by theprocess described herein.

The present inventors have further found that a mixture of a highlydeacetylated chitosan and chitosan oligomers, also forms a hydrogelhaving the desired properties, as described hereinabove.

Thus, according to a further aspect of embodiments of the presentinvention, there is provided a chitosan composition, which forms ahydrogel at near physiological pH and 37° C. the composition comprisingat least one type of a highly deacetylated chitosan having a molecularweight of from 10-4000 KDa (a chitosan polymer) and a degree ofdeacetylation of at least 70%, and at least one type of a chitosanhaving a molecular weight of from 200-20000 Da (namely, a chitosanoligomer), the composition being in a form of an aqueous solution.

The chitosan oligomer can be a highly deacetylated chitosan oligomerhaving a degree of deacetylation of at least 70% and/or a highlyacetylated chitosan oligomer having a degree of acetylation of from 30%to 60%.

In some embodiments, the ratio between the highly deacetylated chitosanpolymer and the chitosan oligomer is higher than 1:1, and can be in therange of from 2:1 to 20:1, depending, inter alia, on the MW of thehighly acetylated chitosan and the chitosan oligomers.

Accordingly, the concentration of the highly acetylated chitosan polymercan be, for example, 1%, 2%, 4%, 10% and any concentration is the rangeof 1-20% (w/w).

The concentration of the chitosan oligomers is selected according to thedesired ratio.

The chitosan oligomers are water-soluble at a pH of 6.5 and higher andthus can also serve as “protectors”, as discussed herein.

Such hydrogels can be utilized in any of the applications describedherein and are prepared as described in Example 8 hereinbelow.

As used herein the term “about” refers to ±10%.

As used herein, the term “pseudo-thermosetting” in connection with thecomposition of the present invention means that temperature does notinduce the gelation of the composition but acts rather as a catalystwhich dramatically shortens the gelation time when risen.

As used herein, the term “neutralized” means a pH of 6.8-7.2.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES Example 1 Preparation of Chitosan Hydrogel

Chitosan with a degree of acetylation of 15% and molecular weight of 65KDa (Koyo, Japan) was dissolved by mixing with 0.9% HCl for 24 hours,forming a type 1 chitosan solution having a chitosan concentration of 3%(w/v).

Homogenously deacetylated chitosan with 51% deacetylation and molecularweight of 220 KDa (Koyo, Japan) was dissolved by mixing with 0.9% HClfor 24 hours, forming a type 2 chitosan solution having a chitosanconcentration of 3% (w/v).

The type 1 and type 2 chitosan solutions were mixed according to thefollowing ratios of type-1 to type-2: 1:1, 1:2 and 1:3, titrated to pH6.8 and left for 24 hours at 4° C., followed by further titration to pH7.2 at 4° C. with sodium hydroxide.

The resulting composition was liquid at room temperature. Uponincreasing the temperature to 37° C. and raising the pH to 7.4, theliquid solution formed a stable semi-solid gel, as illustrated in FIG.1.

Example 2 Degradation Profiles of Chitosan Gels

Pseudo-thermosetting hydrogel (3% w/v) was prepared at a ratio of 1:1w/w of homogenous (type 2) to non-homogenous chitosan (type 1), asdescribed in Example 1 above.

One gram of the gel was placed in 50 ml plastic tubes, in triplicate.

Aliquots of 3 ml of 10% bovine serum media were added to each tube forpredefined times intervals (1, 2, 3, 4, 5, 6, and 7 days). At the end ofeach time interval, the gel was washed 3 times over a period of 24 hoursby repeatedly adding 50 ml of distilled water, leaving at roomtemperature for a few hours and removing the water. The washing processremoved all soluble materials from the gel.

The gel was then frozen, lyophilized and weighed. Weight degradation wascalculated from the change in weight of the samples, as a function oftime interval.

The degradation of the hydrogel by serum enzymes is shown in FIGS. 2 aand 2 b.

Two distinct types of degradation kinetics are shown in FIG. 2 a, a fastphase that terminates within 3-6 days and a slower one that exhibitsonly partial degradation after 14 days. It is believed that the fastphase reflects degradation of type 2 chitosan, which is highly solubleand readily recognized by serum enzymes. The slow kinetic phase isrelated to chitosan type 1 chitosan, which is not readily recognized anddigested by serum enzymes.

Controlling the reacetylation of glucosamine polymer is a very importanttool for manipulating the extent of recognition of the chitosan bylysozyme and consequently for manipulating the rate of hydrogeldegradation. The main factor that controls the activity of the enzyme isthe percentage of N-acetyl glucosamine (NAG) in the polymer (Ran et al.,2005). For this reason decreasing the reacetylation degree from 50% to35% in chitosan Type 2 should allow the rate of degradation to besignificantly decreased, resulting in a much shallower slope. On theother hand, increasing the degree of acetylation of chitosan Type 1should result in faster degradation of the polymer. Selection of theappropriate combination of the two types of chitosans is expected toresult in a single, linear degradation curve over time, instead of thetwo slopes shown in this Figure.

Reference is now made to FIG. 2 b that illustrates mixtures of type 1and type 2 chitosans in ratios of type 1 to type 2 of 1:1, 1:2 and 1:3.As shown in FIG. 2 b, the rate of degradation of the gel is increasedwith increasing ratios of type 2 chitosan to type 1.

Example 3 Slow Release of Proteins by Chitosan Gels

In order to study the potential of the chitosan pseudo thermosettinghydrogel presented herein as a slow release vehicle, hemoglobin andbovine serum albumin (BSA) were used as solutes. These compounds arewell accepted as protein standards.

To one ml solution containing a chitosan mixture as described in Example1 above, in which the final concentration of both chitosans is 3.5%, a25 μl aliquot of BSA or 40 μl of hemoglobin were added, resulting in afinal concentration of 1 mg/ml and 4 mg/ml protein in the hydrogel,respectively. The protein-containing hydrogels were incubated in 3 mlPBS for one week at 37° C. The media was replaced daily, and the amountsof the released protein from the hydrogel was measured, as shown inFIGS. 3-7.

As shown in FIG. 3, in all the tested series, high amounts of hemoglobinwere initially released and the rate of release decreased with time. Noinitial burst was shown.

BSA showed the same profile as hemoglobin (FIGS. 4 and 5). A near linearslope was obtained (FIG. 4). Mixing of the BSA with the gel improved thegel's stability, providing a decreased degradation rate compared to thatof the chitosan mixture alone (FIG. 6).

Comparison of the amounts of the released BSA versus the amounts of thedegraded gel (FIG. 7) showed that the rate of release of the protein wasfaster than the rate of gel degradation.

The data shown in FIG. 7 relate to a single degree of acetylation oftype 1 chitosan and a single molecular weight, which resulted in aprotein release profile having a rate of release which decreased eachday. However, appropriate selection of additional variables such asdegree of reacetylation and molecular weights of the two types ofchitosans allows the characteristics of the gel to be determined, andenables affinity of the protein drug for the chitosan structure to beimproved. Such specific combinations would be expected to provide afixed rate of release of a specific drug, reflecting a combineddiffusion and matrix degradation rate.

Example 4 In vivo Study of Chitosan Gel as Wound Dressing

Psammomys obesus strain rats, which are known to develop diabeticsymptoms when raised in captivity on a high fat diet, were used as amodel of type II Diabetes mellitus. These animals are considered to bean excellent model for simulating chronic skin ulcers of diabetics, andstudy of skin wound healing, due to their tendency to develop profoundinfections, gangrene and sepsis, leading to morbidity and evenmortality.

The following are the common parameters for examining skin ulcershealing:

-   -   1. Timing of neovascularization appearance in the reparative        tissue.    -   2. Reduction in neutrophil activity.    -   3. Accelerated macrophage activity.    -   4. Timing of scar wound closure by a complete        re-epithelialization of the wound.    -   5. Formation of keratinocytes monolayers.    -   6. Binding of the epidermis and the dermis layers by activation        of the fibroblast-depositing extracellular matrix network.

A chitosan-based gel, serving as a biological dressing, was used,avoiding the need for bandaging or suturing, and providing directcoating of the wound bed for enhanced healing. The rate at which varioushealing stages occurred, especially the wound contraction-scar shrinkageand closure stage, was studied over a period of eight days.

The composition of the chitosan mixture includes: FM80 (660 kDa,referred to in Example 7 hereinbelow), DAC-50 (220 kDa, referred to inExample 7 hereinbelow) and a mixture of acetylglucosamine oligomers from1-7 units (Koyo, Osaka, Japan). The formulation includes the threecomponents in a ratio of 1:0.8:0.2, respectively.

Twenty five female Psammomys obesus rats of mature age, each weighing150-160 grams, were used.

Thirteen animals were found to have developed diabetes followingadministration of a high fat diet, starting from 4 to 6 weeks prior today zero.

Six animals having normal euglycemia (normoglycemia), indicatingresistance to development of diabetes upon feeding with a high fat diet,were used as a first control, and six animals with normoglycemia whenfed on a normal low fat, low energy diet, were used as a second control.

At day zero, a round full depth punch biopsy of 6 mm diameter was madethrough the epidermis, dermis and hypodermis to the muscles, at theshaved skin of the neck, using a Metricoconventer-production device.

The injuries of seven diabetic animals were treated by administration ofthe chitosan based-gel of Example 1 to the wound area, while a furthersix animals were left untreated. The gel was reapplied to the wound areaof the treatment group every day.

For a period of seven days all the animals were macro-photographed, andthe dimensions of the wound measured every 3 days. Weight and bloodglucose levels were measured once a week, using digital glucometer,(Ascensia Elite of Bayer), by absorbing a blood drop from a cut createdat the tail of the rat.

After 7 days, the animals were sacrificed and full depth biopsies wereperformed. The skin was collected and placed in fixation solution. Skinsamples were further processed for histological and immuno-histochemicalstaining procedures, to evaluate the differences between treated anduntreated wounds.

Results are shown in FIGS. 8 a and 8 b and FIG. 9. As shown in theseFigures, the treatment group showed a statistically significant increasein wound healing, and a reduction of the period of time required forwound healing, compared to the control group.

Example 5 Rotator Cuff Repair

Rotator cuff tears are a common source of shoulder pain. The incidenceof rotator cuff damage increases with age and is most frequently causedby degeneration of the tendon, rather than injury from sports or trauma.Treatment recommendations vary from rehabilitation to surgical repair ofthe torn tendon(s). The best method of treatment is different for everypatient and indeed many patients do not achieve satisfactory repair oftheir injuries.

The present invention, in some embodiments, overcomes these drawbacks ofthe background art by providing an injectable product allowing deliveryof autologous cells into rotator-cuff tears under ultrasonographiccontrol. In other embodiments, the injectable product allows theincorporation of bone-marrow cells as well, for example for tissuehealing.

Preferably, the procedure is performed as an outpatient procedure or anambulatory procedure requiring local anesthesia.

The initial liquid property of the gel allows full adherence to thetendon tear area.

In vivo experiments were performed on rats for tendon damage and repair(using surgically damaged tendons). The damaged tendons were sutured andwere treated with a chitosan hydrogel as presented herein with bonemarrow cells; the control animals only received sutures. 20 animals werestudied for 3 months. Histologically proven tendon repair and preventionof muscle atrophy were both achieved (results not shown).

Also, in vivo experiments were performed on rats for rotator cuffdamage, again surgically induced. This damage was treated as above.Histological slices of tissue, 6 weeks post surgery, show thatendogenous cells were trapped from the neighboring tissues, improvingthe status of the injured site, compared to non treated control defectsin the contra lateral shoulder. Exemplary results are shown in FIG. 10A(treated) and FIG. 10B (non-treated).

Example 6

A chitosan mixture is designed so as to serve as a reservoir ofnegatively charged substances such as proteoglycans (e.g., chondroitinsulfate), hyaluronate, and/or phospholipids (e.g., phosphatidylcholine). This unique mixture has special rheological propertiessimulating normal synovial fluid, allowing for cartilage regenerationand correction of joint mechanics.

In some embodiments, such a mixture comprises chitosan type 1 andchitosan type 2, as described herein, and further comprises one or morenegatively charged substances, as described herein. In one example, themixture comprises, in addition to the chitosans, phosphatidyl cholineand chondroitin sulfate. In another example, the same mixture isutilized, while further comprising hyaluronate. The latter features aunique mixture of positively and negatively charged polymers, whichprovides to even improved rhelolgical and biological properties of themixture.

A phospholipid-containing chitosan mixture, upon administration, forms ahydrogel composition which can serve as a synovial replacement, for usein, for example, treatment of osteoarthritis.

In order to evaluate the properties of these compositions, the staticcoefficient of friction was measured between 2 cartilage surfacestreated with hyaluronic acid (1%), a chitosan composition comprising 1%chitosan type 1 (having a MW of 660 KDa), 1% of a chitosan type 2(having a MW of 220 KDa) (denoted as chi2gel), and a saline-treatedsystem as a control, as described inhttp://www.pa.uky.edu/˜phy211/Friction_book.html.

FIG. 11 illustrates the model for measuring the static coefficient.

FIG. 12 presents the static friction coefficient between 2 layers ofnormal cartilage, as measured for: no gel (denoted as “saline”); achi2gel as described hereinabove, a chi2gel comprising 0.1% ChondroitinSulfate (denoted cs-4-s), a chi2gel comprising 0.1% Chondroitin Sulfateand 0.1% Phosphatidyl choline (denoted PC 0.1), and a Hyaluronic acid 1%solution (denoted HA 1%).

The obtained data clearly show that combining Chi2Gel with ChondroitinSulfate and Phosphatidyl choline results in a synovial-like frictioncoefficient.

Example 7 Controlling the Conditions for Formation of a ChitosanHydrogel

A chitosan polymer (or oligomer) is defined by its molecular weight, itsdegree of deacetylation, its crystallinity and the mode of distributionof its acetyl groups.

The solubility of chitosan in aqueous solutions is limited. For example,using HCl 0.15N, a chitosan having a MW above 200 KDa is dissolvable atconcentrations lower than 10% (w/v) (higher MW means reduced maximalsolution concentration). HCl 0.15N is a concentration which when fullytitrated becomes the physiological NaCl concentration. Using higherconcentrations of HCl (or other acid like acetic acid) allows higherconcentrations.

The most common commercially available chitosan has a low DA (degree ofacetylation) in the range of 5-30%, and is referred to herein asChit-20. Such a chitosan is a type-1 chitosan, and it precipitates froma solution when at a pH above 6.5. Chit-20 solutions at physiologicalenvironments therefore do not exist, and most of the currently practicedapplications involving implementation of chitosan utilize various typesof solid chitosan (e.g. Procon's gasse).

A unique type of chitosan is a chitosan homogenously deacetylated to 50%or homogenously reacetylated to 30-60% (type 2 chitosan). Such achitosan has a superior solubility in aqueous solutions, as compared tohighly deacetylated chitosans (type 1) and typically remains soluble atneutral and physiological pH, depending on its concentration. Anexemplary such chitosan, having a MW of 220 KDa is referred to herein asChit-50. This type of chitosan, at a concentration of 3% (w/v) or more,forms a gel at pH higher than 7.5.

Gel Formation Using a Mixture of Chitosan Polymers:

When mixing chitosan type 1 (e.g., chit-20) and chitosan type 2 (e.g.,chit-50) at a physiological pH and under certain conditions, noprecipitation of the polymers is observed and instead, the mixture formsa gel. Gel formation involves “coating” (or “protection”) of the chit-20by the chit-50, and is effected by the affinity between the two chitosantypes, which leads to interactions therebetween (e.g., hydrogen bonds,hydrophobic interactions and/or Van der Waals interactions).

The gelation process may be as short as several minutes or as long asmany days and is demonstrated by a gradual yet continuous increase inthe viscosity of the system.

In the conducted (ex-vivo) experiments, gel formation is defined byturning a glass tube that contains the initial solution on its side anddetermining whether the solution flows (or not) and remains stuck to thebottom of the glass tube. Gel formation depends on the type, shape andparameters (e.g., diameter) of tube, as well as the assay time frame.

In this study, gel formation was defined as follows: a one ml solutionwas placed in a 14 mm (in diameter) round-bottomed glass tube and wasincubated at 37° C. overnight. Thereafter, the glass tube was turnedinto a horizontal position and the presence or absence of liquid flowwas determined. Absence of liquid flow indicated that a gel was formed.The gel contains the whole amount of water and remained rigid in a semisolid state. Presence of flow but in a “well distinguished” structurealso indicated that a gel was formed. The presence of liquid flow and/orthe formation of two separate phases, solid and liquid, indicated that agel was not formed.

Controlling Gel Formation:

Materials:

Two commercially available highly deacetylated chitosan polymers(chitosan type 1) were used in this study:

FM80 (MW=660 kDa; 85% DDA (degree of deacetylation); and

FM80S (MW=420 kDa; 91.3% DDA), both obtained from Koyo, Osaka, Japan.

As a highly acetylated chitosan (chitosan type 2), DAC50 (MW=220 kDa;50% DDA), also obtained from Koyo, Osaka, Japan, was used.

Assay Protocol:

Preparation of Stock Solutions: A chitosan polymer (as a powder) wasmixed with HCl 0.15N and the solution was agitated during 24 hours atroom temperature.

The following stock solutions were made:

FM80-2 (2% (w/v) solution of 660 kDa Chit-20) FM80S-2 (2% (w/v) 420 kDaChit-20) DAC50-3 (3% (w/v) 220 kDa Chit-50).

Mixture Formation: the above-described stock solutions, mixtures havinga defined final concentration (w/v) of each chitosan and a defined ratiothereof, were prepared. An exemplary mixture is FM80:DAC50 1.2:1.2, inwhich each of the chitosans are at a final concentrations of 1.2% (w/v),and the ratio therebetween is 1:1.

Titration: The above-described mixtures were slowly titrated, whilebeing cooled in ice water (0° C.), with NaOH (at 2N, 1N and 0.5Nconcentrations), until a pH of about 7.3 was achieved. One ml sampleswere then continuously taken from the mixture during the titration andeach sample was placed in a 14 mm glass tube. The samples were sealedand placed in an incubator at 37° C. overnight.

Gel Formation Testing: Each glass tube was placed in a horizontalposition and gel formation was determined as described hereinabove.

Results:

Table 1 below presents the results obtained for various FM80:DAC50mixtures:

TABLE 1 Mixture Mix Ratio FM80-2 50-3 HCl•15N FM80:DAC50 Gel Formationat various pH values* 3 2 0 1.2:1.2 7.32 7.42 7.5  7.66 N Y Y Y 2.51.667 0.833 1:1 7.35 7.43 7.6  7.7  N N Y Y 2 1.333 1.667 0.8:0.8 7.277.4  7.48 7.54 7.62 N N N N N 1.5 1 2.5 0.6:0.6 7.32 7.43 7.52 7.6  N NN N 2.5 1 1.5  1:0.6 7.4  7.47 7.54 7.61 N N N N *Y = yes, a gel isformed; N = no gel formed

The results show that a mixture of a 660 kDa chitosan type 1, with AD of15% (and similar crystallinity as in FM80) and DAC50 at equal (1:1) w/wratios forms gel at near pH=7.5, when a final concentration of eachchitosan is higher than 0.8% (w/v).

Such a mixture, at final concentrations of 1.2% (w/v) of each chitosan,forms a gel at a wider pH range (below 7.42), as compared to a mixtureat a final concentration of 1% (w/v) of each chitosan (above 7.43), thusindicating that at higher final concentrations the pH range for gelformation is increased (as discussed hereinbelow).

At a FM80:DAC50 ratio higher than 1:1 (e.g., 1:0.6), no gel is formed.

Table 2 below presents the results obtained for various FM80S:DAC50mixtures:

TABLE 2 Mix formula Mix Ratio FM80S-2 50-3 HCl•15N FM80S:DAC50 GelFormation at various pH values* 3 2 0 1.2:1.2 7.37 7.40 7.44 7.50 N Y YY 2.5 1.667 0.833 1:1 7.44 7.53 7.63 N Y Y 9 1.333 1.667 0.8:0.8 7.427.51 7.61 7.7 N N N N 1.5 1 2.5 0.6:0.6 7.44 7.55 7.63 N N N *Y = yes, agel is formed; N = no gel formed

These results further support the findings that a minimal concentrationof each polymer is required in order to achieve gel formation at nearpH=7.5 at the indicated conditions.

This study has shown that parameters influencing gel formation in thetested systems include pH, the relative ratio (w/w) of the chitosantype-1 and type-2 polymers, the molecular weight (MW) of each chitosanpolymer, the final concentration of each chitosan polymer and thetemperature, as follows.

pH: gel formation is pH-dependent, such that solution mixtures form agel only within a certain range of pH. This pH range is increased as thefinal concentrations of the chitosans are increased. The absolute pHvalues increase as the final concentrations of the chitosans decrease.

For example, for a chitosan type 1 (chit-20) having MW of 420 kDa and achitosan type 2 (chit-50) having MW of 220 kDa, each at a concentrationof 1%-1.2% and at a 1:1 w/w ratio, the pH range in which a gel is formedat 4° C. is 7.4-7.7. At higher pH values, precipitation is observed.

It is noted that pH values at 4° C. correlate to pH values lower by 0.5units at 25° C. Thus, pH 7.4 at 4° C. is found to be pH 6.9 at 25° C.

Final Concentration of Chitosan Type 1 (e.g., Chit-20): The chitosantype 1 is the backbone of the formed gel. Hence, gel formation dependson the final concentration of this type of chitosan. It is assumed thatas the MW of the chitosan type 1 increases, the final concentrationdecreases, and vise versa, as the MW decreases the final concentrationincreases.

Molecular Weight of Chitosan Type 1 (e.g., Chit-20): It is assumed thatat higher MW of the highly deacetylated chitosan (type 1), the pHworking range (the range that allows gel formation) decreases, forexample, from 7.4-7.7 for 660 kDa chitosan to 7.0-7.3 for 2,000 kDachitosan. It is further assumed that at higher MW the relativeconcentration of the type 2 chitosan (e.g., Chit-50) required isincreased. It is further assumed that the concentration of type 1chitosan (e.g., chit-20) decreases (e.g., to 0.5%), whereby the pHvalues required for gel formation would shift to 7.2-7.4 (at 4° C.).

It is further assumed that as the molecular weight (MW) chitosan type 1or 2 decreases (e.g., from 420 kDa to 200 kDa), the concentration ofthis chitosan required to form a gel increases. Exemplary suchconcentration is 1.5% (w/v) and higher (with a similar concentration ofthe type 2 chitosan). pH values for gel formation in such conditions areexpected to shift to 7.5-7.8 (at 4° C.).

Concentration of Chitosan Type 2 (e.g., Chit-50): A minimal relativeconcentration of Chit-50 is required for gel formation (e.g., a 1:1ratio). In addition, keeping Chit-20 at constant final concentration andincreasing the concentration of Chit-50 extends the range of otherparameters for gel formation (for example, increases the pH workingrange). Increasing the concentration of Chit-50 further decreases the pHat which a gel is formed.

Molecular Weight of Chitosan Type 2 (e.g., Chit-50): It is expected thatusing Chit-50 having a MW higher than 220 kDa will enable to use areduced relative concentration of Chit-50 in the mixture. Higher MW ofchitosan type 2 results in high protection and improved stability of thechitosan type 1 (having any MW). At such conditions, the pH range forgel formation is expected to increase.

For example, for a 2000 kDa chitosan type 2 with a 660 kDa chitosan type1, pH for gel formation should be about 7.8, and for a 2000 kDa chitosantype 2 and a 2000 kDa chitosan type 1, pH for gel formation should beabout 7.6.

Temperature: The temperature appears to affect the rate of gel formationlinearly. Thus, at 37° C., the gel will form faster than at roomtemperature or at 4° C.

Relative Ratio of Chitosan Type 1 and Type 2: The ratio required for gelformation depends on the MW of each chitosan. For example, as the MW oftype 1 chitosan increases (e.g., to 2000 kDa), its requiredconcentration can be reduced possibly to about 0.5%. However, it isassumed that the ratio between type 2 and type 1 would increase to, forexample, (2:1), 3:1 and even 4:1.

Increasing the MW of type 2 chitosan from 220 kDa to e.g., 2000 kDa, theminimal concentration thereof required for gel formation decreases toe.g., 0.5% (instead of 1%), such that when high MW type 1 chitosan isused, the ratio would be about 1:1.

Example 8 Chitosan Hydrogels Formed from Highly Acetylated and HighlyDeacetylated Chitosan Oligomers and a Highly Deacetylated ChitosanPolymer

Oligomers of highly deacetylated chitosan (e.g., chit-20) do notprecipitate at pH higher than 6.5, as opposed to similar polymers. Thusthe formation of a gel from a mixture of highly deacetylated chitosanoligomers (e.g., MW of 200-20000 Da) and highly deacetylated chitosanpolymers (e.g., MW of 200-2000 KDa) was tested.

Solutions of chit-20 polymers as described in Example 6 hereinabove wereeach mixed with a solution of a chit-20 oligomer (MW=200-1500 Da), thelatter serving in a similar role of “coating” much as chitosan type 2,for protecting the type I chitosan polymer from precipitation. Thetested final concentration of the chit-20 oligomers was 1%, 2%, 4% and10% and the final concentration of the high MW chit-20 polymer (e.g.,FM80) was 1%. The tested ratios (oligomer to polymer) were 1:1; 2:1;4:1; and 10:1.

Except at a 1:1 ratio, all tested mixtures formed a chitosan hydrogel,surprisingly indicating that oligomers of highly deacetylated chitosan(e.g., chit-20) can also provide the required protection for obtainingthe desired hydrogel.

Gel formation was also observed with FM80s (MW of 420 kDa) and highlydeacetylated chitosan oligomers having MW of (MW=200-2000 Da), at aratio of 1:3.

Similar results, namely gel formation, are obtained for mixtures ofhighly deacetylated chitosan polymers (e.g., MW of 200-2000 KDa) andhighly acetylated chitosan oligomers (e.g., chit-50 oligomers having MWof 1000-20000 Da).

Example 9 Intraarticular Injection of a Chitosan Composition ComprisingHyaluronate Delays Osteoarthritis Progression and Reduces Pain in a RatMeniscectomy Model

Animal Models and Procedures.

Wistar rats of 0.3-kilogram-weight male rats were used in the study.Knee osteoarthritis occurs predictably after partial medialmeniscectomy. The disease develops in a time-dependent and predictablefashion. It is a common model assessing the effect of antiosteoarthritisdrugs. General anesthesia was induced by Ketamine 80 mg/kg and Xylazine8 mg/kg. In the right knee, 200-microliters of 2% (w/v)chitosan-hyaluronate hybrid gel was injected at the time of meniscectomyin 9 animals and two week after meniscectomy in another four animals.The contralateral knee served as control to either saline or hyaluronate(1% gel 200-microliters, produced by Savient Pharmaceuticals, Inc., EastBrunswick, N.J., USA) injection. The rats were allowed unrestrictedmotion after the surgery and evaluated every six weeks under imageintensification. The following parameters were evaluated: degree ofmedial joint space opening and unloaded joint space width. After 3months, the animals were sacrificed, and histological examination wasperformed. Animal knees were randomized after incapacitance testingdemonstrated similar weight-bearing on both hindlimbs. In one knee,either saline (in 4 animals) or hyaluronate was injected (5 animals). Inthe contralateral knee, chitosan-hyaluronate mixture was injected. Inanother four animals, the injection was performed under generalanesthesia one week after meniscectomy.

Chitosan-Hyaluronate Hybrid Gel.

A chitosan hyaluronic acid hybrid has been used. Briefly, a mixture ofchitosans and oligochitin (FM80, DAC50, and oligochitin from Koyochemicals Ltd., Japan) was solubilized in HCl 0.13N and titrated withsodium hydroxide to pH of between 6.6 to 7.2. This was followed by anaddition of hyaluronic acid (molecular weight of 3 million Dalton,Ferring Ltd.). Genepin (Challenge Bioproducts Co., Ltd., Taiwan), anatural cross-linker is added to the chitosan hyaluronic acidcomposition at 0.2% (w/v) just before injecting in order to acceleratethe gelation.

Incapacitance Tester Evaluation.

Incapacitance tester is a device allowing assessing changes in hind pawweight distribution between the right (osteoarthritic) and left(contralateral control) limbs. It has been utilized as an index of jointdiscomfort and may be useful for the discovery of novel pharmacologicagents in human osteoarthritis. The animals were assessed prior tosurgery as well as 24 hours after surgery for the relative amount ofweight bearing on either knee using a Linton Incapacitance meter (LintonInstrumen-tation, Norfolk, UK). The animals were ranked according to thedifference between the right limb and the left limb weight bearing. Theexperimental versus control knees were then determined so that there wassimilar distribution of right-limbed versus left-limbed animals. Thisstep is important in order to prevent bias related to animal“handedness.” Animals were examined again two weeks after injection ofthe intra-articular therapy. The animals were examined again 14 daysafter instillation of the intra-articular therapeutic agent.

Histological Evaluation.

The rats were euthanized using intraperitoneal 200 mg/kg sodiumpentobarbital injection. The knees were dissected out and processed forroutine histology following fixation with 1% cetylpyridinium chloride—4%formalin solution for 48 hours. Decalcification was carried out in EDTAfor three weeks on average. Masson's trichrome and hematoxylin stainswere evaluated. The following parameters were measured: cartilagethickness at the lowest part of the medial femoral condyle, osteophyteformation, cyst formation, and subchondral bone plate thickness.Quantitative histology was performed using an image analysis program(ImageJ). Statistical analysis was performed using the Microsoft Exceladd-in program Analyze-it version 2.22.

Results

All animals survived the surgery, and their joint did not exhibit anyevidence of inflammation or wound breakdown. Weight gain proceeded asexpected. Incapacitance Tester Evaluation revealed that the differencebetween the experimental and control knee averaged 1±2 grams prior tosurgery. The relative weight-bearing did not significantly changedfollowing meniscectomy (2±2). After 2 weeks, the amount of weightbearing was measured again. The animals bore weight preferentially onthe experimental knee (16.6±4 grams). This difference was found to besignificant as compared to the measurement 24 hours following surgery(Student's t-test, P<0.017).

Histological Evaluation revealed that cartilage thickness wassignificantly (Student's t-test. P<0.043) increased in the experimentalgroups (170±8) as compared with the control knees (108±10) (FIG. 13).

The hybrid gel appears to undergo self-assembly perhaps due tohyaluronate molecules aligning the smaller chitosan molecules (FIG. 14)and does not seem to induce an inflammatory response when injectedsubcutaneously in rats (FIG. 15). Cyst grading was performed using a4-point scale, wherein means no cyst; 1 means minimal cyst; 2 meanslarge cyst; and 3 means very large cyst. There were no cysts formed inthe experimental group (average grade 0), while in the control group theaverage was 0.55±0.5. This difference was found to be significant(Student's t-test, P<0.047).

Subchondral bone plate thickness results showed no significantdifference between the groups. Osteophyte grading was performed using a4-point scale, wherein 0 means no osteophyte; 1 means minimalosteophyte; 2 means large soft tissue osteophyte; and 3 means large bonyosteophyte. Average grade in the chitosan group was 0.8±0.5, while inthe control group it was 1.2±0.3.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification and in addition, U.S. Pat. No. 8,153,612, are hereinincorporated in their entirety by reference into the specification, tothe same extent as if each individual publication, patent or patentapplication was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting.

What is claimed is:
 1. A chitosan composition which forms a hydrogel atnear physiological pH and at 37° C., comprising at least one acetylatedchitosan having a degree of acetylation in the range of from about 30%to about 60%, and at least one deacetylated chitosan having a degree ofdeacetylation that is about 70% to about 95%, the composition being inthe form of an aqueous solution at neutral pH, wherein the ratio of saidacetylated chitosan to said deacetylated chitosan in the composition isin the range of 1:1 to 4:1.
 2. The chitosan composition of claim 1,further comprising at least one negatively charged polysaccharide. 3.The chitosan composition of claim 2, wherein the negatively chargedpolysaccharide is hyaluronic acid.
 4. The chitosan composition of claim1, further comprising acetylglucosamine oligomers.
 5. The chitosancomposition of claim 4, wherein the acetylglucosamine oligomers compriseTri-N-acetyl-glucosamine.
 6. The chitosan composition of claim 4,wherein the acetylglucosamine oligomers are present in the compositionat a concentration of between 0.05% to 10% w/v.
 7. The chitosancomposition of claim 1, further comprising a cross-linking agent in anamount sufficient to accelerate gelation of the chitosan composition. 8.The chitosan composition of claim 7, wherein said cross-linking agent iscapable of cross-linking the amine or hydroxyl residues of thechitosans.
 9. The chitosan composition of claim 7, wherein said amountof the cross linking agent is in the range of between 0.1% to 2% w/v.10. The chitosan composition of claim 7, wherein the cross-linking agentis added to the chitosan composition prior to introducing said chitosancomposition into a site in vivo.
 11. The chitosan composition of claim7, wherein the cross-linking agent is genipin.
 12. A process forpreparing a stable pH-dependent and temperature-dependent chitosanhydrogel composition comprised of at least one acetylated chitosanhaving a degree of acetylation of from about 30% to about 60%, and atleast one deacetylated chitosan having a degree of deacetylation that isabout 70% to about 95%, which process comprises: d) dissolving saidacetylated chitosan and said deacetylated chitosan in an acidic aqueoussolution at a temperature between 0° C. to 10° C., to thereby form acomposite solution, wherein the ratio of said acetylated chitosan andsaid deacetylated chitosan in said composite solution is in the range of1:1 to 4:1; e) adjusting the pH of said composite solution to a value of6.6 to 7.4 at a temperature between 0° C. to 10° C.; and f) increasingat least one of the temperature of said composite solution to about 37°C. and/or raising the pH to physiological pH.
 13. The process of claim12, further comprising adding a negatively charged polysaccharide to thecomposition before step c.
 14. The process of claim 13 wherein thepolysaccharide is hyaluronic acid.
 15. The process of claim 12, furthercomprising adding acetylglucosamine oligomers to the composition. 16.The process of claim 12, further comprising adding a cross-linking agentin an amount sufficient to accelerate gelation of the chitosancomposition, wherein the cross-linking agent is added to the compositionprior to introducing said chitosan composition into a site in vivo. 17.The process of claim 16 wherein the cross-linking agent is genipin. 18.A hydrogel chitosan composition formed by the process of claim
 12. 19. Amethod of treating medical condition in a subject in need thereof,comprising applying into a site of the medical condition a chitosancomposition comprising at least one acetylated chitosan having a degreeof acetylation in the range of from about 30% to about 60%, and at leastone deacetylated chitosan having a degree of deacetylation that is about70% to about 95%, the composition being in the form of an aqueoussolution at neutral pH, wherein the ratio of said acetylated chitosan tosaid deacetylated chitosan in the solution is in the range of 1:1 to4:1.
 20. The method of claim 19, wherein the medical condition isselected from the group consisting of: osteoarthritis, fracture repair,bone structural support, cartilage repair, intervertebral disc repair,meniscal repair, bone reconstruction, bone filling and synovial fluidreplacement.
 21. A kit for producing a chitosan hybrid hydrogel,comprising: (i) a container containing a chitosan composition comprisingat least one acetylated chitosan having a degree of acetylation in therange of from about 30% to about 60%, and at least one deacetylatedchitosan having a degree of deacetylation that is about 70% to about95%, wherein the ratio of said acetylated chitosan to said deacetylatedchitosan in the solution is in the range of 1:1 to 4:1; (ii) a containercontaining a cross-linking agent in an amount sufficient to accelerategelation of the chitosans; and (iii) instructions for preparing thechitosan hybrid hydrogel.
 22. The kit of claim 21, wherein the containercontaining a chitosan composition further comprises a negatively chargedpolysaccharide.
 23. The kit of claim 22, wherein the polysaccharide ishyaluronic acid.
 24. The kit of claim 21, wherein the containercontaining a chitosan composition further comprises acetylglucosamineoligomers.
 25. The kit of claim 24, wherein the acetyl glucosamineoligomers comprise Tri-N-acetyl-glucosamine.
 26. The kit of claim 21,wherein the kit further comprises a container containing a solvent fordissolving the cross-linking agent.